Green Fluorescent ProteinGreen Fluorescent Protein

Molecular GeneticsMolecular Genetics

Green Fluorescent ProteinGreen Fluorescent Protein

Green Fluorescent Protein (GFP) has existed for more than one hundred and sixty million years in one species of jellyfish, Aequorea victoria

Green Fluorescent Protein (GFP) has existed for more than one hundred and sixty million years in one species of jellyfish, Aequorea victoria

FluorescenceFluorescence

Wild type GFP from jellyfish has two excitation peaks, a major one at 395 nm and a minor one at 475 nm with extinction coefficient of 30,000 and 7,000 M-1 cm-1, respectively. Its emission peak is at 509 nm in the lower green portion of the visible spectrum.

Wild type GFP from jellyfish has two excitation peaks, a major one at 395 nm and a minor one at 475 nm with extinction coefficient of 30,000 and 7,000 M-1 cm-1, respectively. Its emission peak is at 509 nm in the lower green portion of the visible spectrum.

FluorophoreFluorophore

Eleven strands on the outside of cylinders form the walls of the structure. The cylinders have a diameter of 30A and a length of 40A.

Small sections of alpha-helix form caps on the ends of the cylinders and an irregular alpha-helical segment also provide a scaffold for the fluorophore which is located in the geometric center of the cylinder. The strands of beta-sheet are tightly fitted to each other like staves in a barrel.

Eleven strands on the outside of cylinders form the walls of the structure. The cylinders have a diameter of 30A and a length of 40A.

Small sections of alpha-helix form caps on the ends of the cylinders and an irregular alpha-helical segment also provide a scaffold for the fluorophore which is located in the geometric center of the cylinder. The strands of beta-sheet are tightly fitted to each other like staves in a barrel.

FluorophoreFluorophore The fluorophore itself is a p-

hydroxybenzylidene-imidazolidone. It consists of residues Ser65- dehydroTyr66 - Gly67 of the protein. The cyclized backbone of these residues forms the imidazolidone ring.

The fluorescence is not an intrinsic property of the Ser-Tyr-Gly tripeptide. The amino acid sequence Ser-Tyr-Gly can be found in a number of other proteins as well.

This peptide is neither cyclized in any of these, nor is the tyrosine oxidized. None of these proteins has the fluorescence of GFP.

The fluorophore itself is a p-hydroxybenzylidene-imidazolidone. It consists of residues Ser65- dehydroTyr66 - Gly67 of the protein. The cyclized backbone of these residues forms the imidazolidone ring.

The fluorescence is not an intrinsic property of the Ser-Tyr-Gly tripeptide. The amino acid sequence Ser-Tyr-Gly can be found in a number of other proteins as well.

This peptide is neither cyclized in any of these, nor is the tyrosine oxidized. None of these proteins has the fluorescence of GFP.

Absorption spectrum of gfp

Absorption spectrum of gfp

Excitation and EmissionExcitation and Emission

Amino acid Sequencegfp

Amino acid Sequencegfp

1 mskgeelftg vvpilveldg dvnghkfsvs gegegdatyg kltlkfictt gklpvpwptl

61 vttfsygvqc fsrypdhmkq hdffksampe gyvqertiff kddgnyktra evkfegdtlv

121 nrielkgidf kedgnilghk leynynshnv yimadkqkng ikvnfkirhn iedgsvqlad

181 hyqqntpigd gpvllpdnhy lstqsalskd pnekrdhmvl lefvtaagit hgmdelyk//

1 mskgeelftg vvpilveldg dvnghkfsvs gegegdatyg kltlkfictt gklpvpwptl

61 vttfsygvqc fsrypdhmkq hdffksampe gyvqertiff kddgnyktra evkfegdtlv

121 nrielkgidf kedgnilghk leynynshnv yimadkqkng ikvnfkirhn iedgsvqlad

181 hyqqntpigd gpvllpdnhy lstqsalskd pnekrdhmvl lefvtaagit hgmdelyk//

Blue Fluorescent ProteinBlue Fluorescent Protein

1 mskgeelftg vvpilveldg dvnghkfsvs gegegdatyg kltlkfictt gklpvpwptl

61 vttfxvqcfs rypdhmkrhd ffksampegy vqertiffkd dgnyktraev kfegdtlvnr

121 ielkgidfke dgnilghkle ynfnshnvyi madkqkngik vnfkirhnie dgsvqladhy

181 qqntpigdgp vllpdnhyls tqsalskdpn ekrdhmvlle fvtaagithg mdelyk

1 mskgeelftg vvpilveldg dvnghkfsvs gegegdatyg kltlkfictt gklpvpwptl

61 vttfxvqcfs rypdhmkrhd ffksampegy vqertiffkd dgnyktraev kfegdtlvnr

121 ielkgidfke dgnilghkle ynfnshnvyi madkqkngik vnfkirhnie dgsvqladhy

181 qqntpigdgp vllpdnhyls tqsalskdpn ekrdhmvlle fvtaagithg mdelyk

Blue Fluorescent ProteinBlue Fluorescent Protein

Blue fluorescent protein is a variant of the GFP with a Hit to Tyr substitution at position 66 and a second substitution from Tyr to Phe at position 145.

Blue fluorescent protein is a variant of the GFP with a Hit to Tyr substitution at position 66 and a second substitution from Tyr to Phe at position 145.

ChameleonsChameleons

Different mutations causes different colors

Different mutations causes different colors

Fluorescence in NatureFluorescence in Nature

Fluorescent Molecules used in research

Fluorescent Molecules used in research

Fluorescence in ResearchFluorescence in Research

DNA TransformationDNA Transformation

Uptake of naked DNA molecule from the environment and incorporation into recipient in a heritable form

Competent cellcapable of taking up DNA

May be important route of genetic exchange in nature

Uptake of naked DNA molecule from the environment and incorporation into recipient in a heritable form

Competent cellcapable of taking up DNA

May be important route of genetic exchange in nature

DNA bindingprotein

nuclease – nicks and degrades onestrand

competence-specificprotein

Streptococcus pneumoniae

Bacteria and transformationBacteria and

transformation Not all bacteria

can be transformed in nature

Streptococcus pneumonia, Haemophilus influenza, and Neisseria gonorrhea

Not all bacteria can be transformed in nature

Streptococcus pneumonia, Haemophilus influenza, and Neisseria gonorrhea

Transformationhttp://www.dnalc.org/ddnalc/resources/transformation2.html

Transformationhttp://www.dnalc.org/ddnalc/resources/transformation2.html

Uptake of DNA can only occur at a certain cell density

Cells need to be in the log phase of growth

A competence factor is required for the uptake of DNA from the environment

Uptake of DNA can only occur at a certain cell density

Cells need to be in the log phase of growth

A competence factor is required for the uptake of DNA from the environment

Genetic recombination and transformation in the

laboratory

Genetic recombination and transformation in the

laboratory Plasmids are designed to

contain genes of interest Transformation done in

laboratory with species that are not normally competent (E. coli)

Variety of techniques used to make cells temporarily competent calcium chloride treatment

makes cells more permeable to DNA

Plasmids are designed to contain genes of interest

Transformation done in laboratory with species that are not normally competent (E. coli)

Variety of techniques used to make cells temporarily competent calcium chloride treatment

makes cells more permeable to DNA

Cloning vectorsCloning vectors

pGlo and transformationpGlo and transformation

Lab protocolLab protocol Obtain two tubes containing CaCl2 Label One tube +DNA, Label the other tube –

DNA/Group These tubes have been on ice for one hour+ Add your bacteria cells and incubate for thirty Pick bacterial colonies or cells and add them to both the

+ and – tubes Vortex the tube and replace on ice To the + tube add plasmid DNA 10 ul of either green or blue 5ul of blue and green Do not add plasmid to the – DNA tube Check tips to make sure that you added the plasmid to your cells Mix by pulsing in the microcentrifuge

Obtain two tubes containing CaCl2 Label One tube +DNA, Label the other tube –

DNA/Group These tubes have been on ice for one hour+ Add your bacteria cells and incubate for thirty Pick bacterial colonies or cells and add them to both the

+ and – tubes Vortex the tube and replace on ice To the + tube add plasmid DNA 10 ul of either green or blue 5ul of blue and green Do not add plasmid to the – DNA tube Check tips to make sure that you added the plasmid to your cells Mix by pulsing in the microcentrifuge

Heat shockHeat shock

Incubate for thirty minutes on iceKeep your tubes in ice in a cup and

go to water bathHeat shock at 42oC for 90 secondsRemove tubes from bath and

immediately place back on ice for 2 minutes

Incubate for thirty minutes on iceKeep your tubes in ice in a cup and

go to water bathHeat shock at 42oC for 90 secondsRemove tubes from bath and

immediately place back on ice for 2 minutes

RecoveryRecovery

Add 250 ul of the Luria Broth to the transformation tubes. The Luria Broth is rpewarmed. It should incubate for at least fifteen minutes at 37oC. Place your tubes in the incubator.

Add 250 ul of the Luria Broth to the transformation tubes. The Luria Broth is rpewarmed. It should incubate for at least fifteen minutes at 37oC. Place your tubes in the incubator.

ProtocolProtocol

Preparation of platesTwo plates should be labeled + DNA+DNA LB

+DNA-LB’AMPTwo plates should be labeled – DNA

as aboveAdd 250 ul of transforming solution

and Luria to each plate

Preparation of platesTwo plates should be labeled + DNA+DNA LB

+DNA-LB’AMPTwo plates should be labeled – DNA

as aboveAdd 250 ul of transforming solution

and Luria to each plate

Spread PlatesSpread Plates

Make a spread plate by spreading the 250 ul of sample first horizontally, then vertically , and finally diagonally.

Stack plates and tape. Let plates sit bottom side down until fluid is absorbed into the agar.

Make a spread plate by spreading the 250 ul of sample first horizontally, then vertically , and finally diagonally.

Stack plates and tape. Let plates sit bottom side down until fluid is absorbed into the agar.

Incubate overnightIncubate overnight

Incubate overnight at 37oC.Check for growthCheck selection plates for

transformants Use the long range uv light to

check for fluorescence.

Incubate overnight at 37oC.Check for growthCheck selection plates for

transformants Use the long range uv light to

check for fluorescence.